1. Field of the Invention
The present invention relates to assays for predicting the chemosensitivity of tissues to particular drugs, and more specifically to a method for measuring both the anti-migratory effect and cytotoxic effect of drugs on tissues in vitro.
2. Description of Prior Art
Solid tissue cancers are broadly defined by two general characteristics, namely: (1) a mass of hyperproliferating cells of clonal origin, and (2) acquisition of an aggressively invasive phenotype, wherein cancer cells leave the tissue of origin and establish new tumor metastases at distant sites. Current methods for evaluating the effect of accepted and experimental anti-cancer drugs on human cancers focus on measuring the arrest of hyperproliferation or the stimulation of cell death, both markers of only the first characteristic of cancer.
In general, these current methods require that single cells be isolated from a tumor biopsy and then grown in a culture. The cultured cells are then exposed to a drug, and after a certain amount of time has elapsed cytostatic or cytotoxic effects are measured. These measurements are made in many ways, including: incorporation of the toxic substance, inducement of programmed cell death (termed apoptosis), depletion of intracellular metabolites such as adenosine triphosphate (ATP), depression of cell growth, and compromise of the cell membrane.
The most significant drawback of these methods is the inability to assess the effect of anticancer drugs on the second, and most lethal, characteristic of cancers, i.e., the invasive characteristic that leads to establishment of tumors at distant sites. Other drawbacks include:
1. The requirement to grow/expand biopsy cells in culture before testing allows selective expansion of subsets of tumor cells that are not necessarily representative of the entire tumor. In addition, many biopsies suffer from low plating efficiency, precluding them from being tested.
2. Measurements of cellular metabolism cannot discriminate cells that are injured and will recover from those which are truly dead.
3. Assays using differential staining techniques are subject to individual interpretation, and frequently cannot distinguish live cells from cellular debris.
4. All assays that measure a single biochemical endpoint, such as cell proliferation or inhibition of DNA synthesis are limited by the particular characteristics of that endpoint, and do not accurately reflect cell survival.
5. These assays suffer from long turn-around time, ranging from 5 days to several weeks.
It is therefore desirable to provide an alternative assay for measuring the chemosensitivity of cancer biopsies that does not require the expansion of cells in culture, can assess the effect of anticancer drugs on both the hyperproliferation and invasive characteristics of human cancers, that is not dependent on cell metabolism or differential staining, and can be performed quickly with a minimum of manipulations.
Alternative assays proposed to predict the chemosensitivity of biopsies in vitro can be found in U.S. Pat. Nos. 4,816,395 and 4,937,182. A review of several chemosensitivity assays that use metabolite production and hyperproliferation analysis as chemosensitivity determinants is given by Bellamy, W. T., Prediction of response to drug therapy of cancer. A review of in vitro assays. Drugs 44(5): pp 690-708, (1992), Sevin, B. U., et al., Current status and future directions of chemosensitivity testing. Contrib Gynecol Obstet 19: pp 179-194, (1994), and Cramer, A. B., et al., Chemosensitivity testing: a critical review. Crit Rev Clin Lab Sci 28(5-6): pp 405-413 (1991, Hoffman, R. M., In vitro assays for chemotherapy sensitivity. Crit Rev Oncol Hematol 15(2): pp 99-111, (1993). A chemosensitivity assay using radioactive nucleotide incorporation is given by Kitaoka A., et al, Improvement of in vitro chemosensitivity assay for human solid tumors by application of a preculture using collagen matrix. Clin Cancer Res 3(2): pp 295-299, (1997). A method using DNA synthesis as an indicator of cell growth is given by Kawabata, K. et al., Anticancer chemosensitivity and growth rate of freshly separated human colorectal cancer cells assessed by in vitro DNA synthesis inhibition assay. Anticancer Res 18(3A): pp 1633-1640 (1998). Chemosensitivity assays that measure cell viability by cellular metabolism are: Furukawa, T., et al., Clinical applications of the histoculture drug response assay. Clin Cancer Res 1(3): pp 305-311, (1995), and Kawamura, H., et al., The usefulness of the ATP assay with serum-free culture for chemosensitivity testing of gastrointestinal cancer. Eur J Cancer 33(6): pp 960-966, (1997). An embodiment of the present invention is described in Rust, W. L., Screening assay for promigratory/antimigratory compounds. Anal Biochem 280(1) pp 11-19, (2000). Each of these suffers from at least one of the disadvantages listed above.
The present invention takes advantage of several aspects of a procedure for automatically measuring cell migration described in U.S. Pat. No. 5,601,997 to Tchao, modified and adapted for the novel use described herein. The present invention further incorporates for novel use the cell death determination procedure described by Nieminen, A. L., A novel cytotoxicity screening assay using a multiwell fluorescence scanner. Toxicol Appl Pharmacol 115(2): pp 147-155, (1992).
The present invention provides the first efficient method for simultaneously measuring the anti-migratory effect and cytotoxic effect of drugs on biopsy cells without the need to expand the cells in culture. This assay is particularly useful for predicting the chemosensitivity of an individual patient""s cancer to therapeutic drugs. This assay is unique among chemosensitivity assays for the ability to measure both the anti-migratory and cytotoxic effects of drugs. Because the formation of solid tissue cancer is dependent upon the invading cell""s ability to migrate across tissues as well as hyperproliferation, a chemosensitivity assay that measures the both the anti-migratory and cytotoxic effect of drugs is a more comprehensive and more sensitive method for determining the chemosensitivity of biopsies than other assays. Moreover, obviating the need to expand cells in culture reduces the risk of experimental factors which can bias the assay outcome, and allows the assay to be performed more quickly than current methods.
In accordance with the present invention biopsy samples are dissociated into individual cells which are exposed to anticancer drugs and introduced into a top chamber of a migration assay apparatus. A stimulant then induces the cells to migrate across a porous membrane. After a period of time, migrated cells are labeled with a live cell fluorescent indicator and non-migrated cells are labeled with a fluorescent indicator of cell death. The fluorescence of both fluorophores are measured in a fluorescence plate reader. The fluorescence intensity of the cell death reporter indicates the sensitivity of the cells to cytotoxic effects of the drugs. The fluorescence emitted from the migrated cells indicates ability of the biopsy cells to migrate in the presence of the drugs.
Significantly, the present invention is distinct from all the prior art, including the procedure described in U.S. Pat. No. 5,601,997 to Tchao, in that the prior art procedures all require that the cells in the assay be first pre-labeled. The presence of a labeling chemical dye in the prior art procedures can influence migration behavior and may interact with the chemical agent being tested. In contradistinction, the inventive assay does not introduce chemical agents into the process, as it is not intended to for kinetic analysis of the migratory behavior of cells, and the incorporation of a cytotoxic assay into the procedure is entirely novel.